U.S. patent number 5,401,516 [Application Number 08/051,739] was granted by the patent office on 1995-03-28 for modified hydrolyzed vegetable protein microspheres and methods for preparation and use thereof.
This patent grant is currently assigned to Emisphere Technologies, Inc.. Invention is credited to Evgueni N. Barantsevitch, Sam J. Milstein.
United States Patent |
5,401,516 |
Milstein , et al. |
March 28, 1995 |
Modified hydrolyzed vegetable protein microspheres and methods for
preparation and use thereof
Abstract
Modified hydrolyzed vegetable protein microspheres and methods
for their preparation and use as oral delivery systems for
pharmaceutical agents are described.
Inventors: |
Milstein; Sam J. (Larchmont,
NY), Barantsevitch; Evgueni N. (New Rochelle, NY) |
Assignee: |
Emisphere Technologies, Inc.
(Hawthorne, NY)
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Family
ID: |
26729780 |
Appl.
No.: |
08/051,739 |
Filed: |
April 22, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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995508 |
Dec 21, 1992 |
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Current U.S.
Class: |
424/491; 424/489;
424/498; 424/426 |
Current CPC
Class: |
A61K
9/1658 (20130101) |
Current International
Class: |
A61K
9/16 (20060101); A61K 009/16 () |
Field of
Search: |
;424/491,426,489,498 |
References Cited
[Referenced By]
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Primary Examiner: Page; Thurman K.
Assistant Examiner: Benston, Jr.; William E.
Attorney, Agent or Firm: Darby & Darby
Parent Case Text
This is a continuation-in-part of U.S. patent application Ser. No.
07/995,508, filed Dec. 21, 1992, now abandoned. This invention
relates to modified hydrolyzed vegetable proteins and microspheres
made from them. The microspheres releasably encapsulate active
agents and are suitable for oral administration to mammals. Methods
for the preparation of such microspheres are also disclosed.
Claims
What is claimed is:
1. A method for preparing a modified hydrolyzed vegetable protein
microsphere comprising the steps of:
(a) mixing a hydrolyzed vegetable protein with an aqueous alkaline
solution to form a solution;
(b) treating said solution with an amine reactive agent to form a
mixture containing a modified hydrolyzed vegetable protein;
(c) recovering said modified hydrolyzed vegetable protein;
(d) incubating said modified hydrolyzed vegetable protein in a
aqueous acid solution to form said modified hydrolyzed vegetable
protein microspheres; and
(e) collecting said protein microspheres.
2. The method according to claim 1, wherein said aqueous alkaline
solution comprises potassium or sodium hydroxide.
3. The method according to claim 1, wherein step (a) is performed
at a temperature ranging between about 35.degree. C. and about
40.degree. C.
4. The method according to claim 1, wherein said amine reactive
agent is selected from the group consisting of benzene sulfonyl
chloride and benzoyl chloride.
5. The method according to claim 4, wherein said aqueous acid
solution further comprises a biologically-active agent.
6. The method according to claim 5, wherein said
biologically-active agent comprises a monoclonal antibody, insulin,
calcitonin, or erythropoietin.
7. The method according to claim 13, wherein said microspheres are
hollow and said biologically-active agent is encapsulated
therein.
8. The method according to claim 5, wherein said
biologically-active agent is a protein.
9. The method according to claim 8, wherein said protein is
insulin.
10. The method according to claim 8, wherein said protein is human
growth factor.
11. The method according to claim 8, wherein said protein is bovine
growth factor.
12. The method according to claim 8, wherein said protein is
interferon.
13. The method according to claim 8, wherein said protein is
interleukin II.
14. The method according to claim 8, wherein said protein is
atrial
15. The method according to claim 8, wherein said protein is
calcitonin.
16. The method according to claim 5, wherein the biologically
active agent is a polysaccharide.
17. The method according to claim 16, wherein said polysaccharide
is heparin.
18. The method according to claim 5, wherein said biologically
active agent is an antigen.
19. The method according to claim 5, wherein said biologically
active agent is a monoclonal antibody.
20. The method according to claim 5, wherein said biologically
active agent is aspirin.
21. The method according to claim 5, wherein said biologically
active agent is erythropoietin.
22. The method according to claim 1, wherein said biologically
active agent as a NSAID.
23. The method according to claim 1, wherein the active agent is an
antimicrobial agent.
24. The method according to claim 1, wherein the active agent is
quinolone.
Description
BACKGROUND OF THE INVENTION
The available means for delivering pharmaceutical and therapeutic
agents to mammals often are severely limited by chemical or
physical barriers or both, which are imposed by the body. For
example, oral delivery of many biologically-active agents would be
the route of choice if not for the presence of chemical and
physicochemical barriers such as extreme pH in the gut, exposure to
powerful digestive enzymes, and impermeability of gastrointestinal
membranes to the active ingredient. Among the numerous
pharmacological agents which are known to be unsuitable for oral
administration are biologically active peptides and proteins, such
as insulin. These agents are rapidly destroyed in the gut by acid
hydrolysis and/or by proteolytic enzymes.
Much research has been devoted to developing effective oral drug
delivery methods and systems for these vulnerable pharmacological
agents. The proposed solutions have included:
(a) co-administration of adjuvants (such as resorcinols and
non-ionic surfactants polyoxyethylene oleyl ether and n-hexadecyl
polyethylene ether to increase the permeability of the intestinal
walls; and
(b) co-administration of enzymatic inhibitors, such as pancreatic
trypsin inhibitor, diisopropylfluorophosphate (DFF) and trasylol to
avoid enzymatic degradation.
The use of such substances, in drug delivery systems, is limited
however either because of their:
(a) inherent toxicity when employed in effective amounts; or
(b) failure to protect the active ingredient or promote its
absorption; or
(c) adverse interaction with the drug.
Liposomes as drug delivery systems have also been described. They
provide a layer of lipid around the encapsulated pharmacological
agent. The use of liposomes containing heparin is disclosed in U.S.
Pat. No. 4,239,754 and several studies have been directed to the
use of liposomes containing insulin; e.g., Patel et al. (1976) FEBS
Letters Vol. 62, page 60 and Hashimoto et al. (1979) Endocrinol.
Japan, Vol. 26, page 337. The use of liposomes, however, is still
in the development stage and there are continuing problems,
including:
(a) poor stability;
(b) inadequate shelf life;
(c) limited to low MW (<30,000) cargoes;
(d) difficulty in manufacturing;
(e) adverse interactions with cargoes.
More recently, artificial amino acid polymers or proteinolds,
forming microspheres, have been described for encapsulating
pharmaceuticals. For example, U.S. Pat. No. 4,925,673 (the '673
patent), the disclosure of which is hereby incorporated by
reference in its entirety, describes such microsphere constructs as
well as methods for their preparation and use. The microspheres of
the '673 patent are useful for encapsulating a number of active
agent's, however there is a need in the art for microsphere
carriers that can encapsulate a broader range of active agents such
as lipophilic drugs.
Additionally, the method employed in the '673 patent for preparing
proteinoids results in a complex mixture of high molecular weight
(MW) (>1000 daltons) and low MW (.ltoreq.1000 daltons)
peptide-like polymers which are difficult to separate. Moreover,
small amounts of the low MW microsphere-forming proteinoids are
obtained. Thus, an improved method of preparing low molecular
weight sphere-forming proteinoids is also desired.
SUMMARY OF THE INVENTION
The present invention relates to a modified hydrolyzed vegetable
protein microsphere and to a method for preparation of such
microspheres. The invention provides stable microspheres which are
preparable from inexpensive hydrolyzed vegetable protein, e.g.
soybean protein, and a simple and economical method for making such
microsphere. Microspheres made according to the invention display
improved stability and performance in delivering biologically
active materials to mammals.
According to the invention, modified hydrolyzed vegetable
microspheres are prepared by dissolving hydrolyzed vegetable
protein in an aqueous alkaline solution and adding a chemical
modifier which reacts with free amine residues present in the
hydrolyzed protein. The pH of the reaction mixture is then lowered
until the modified vegetable protein precipitates out from the
mixture. The recovered protein readily forms microspheres and can
be used to encapsulate various cargoes such as pharmaceutical
agents. The microspheres are non-toxic and can be orally or
parenterally administered to mammals.
DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates levels of glucose detected in rat serum taken
from rats orally administered microsphere encapsulated insulin or
raw (unencapsulated) insulin as described in Example 4.
FIG. 2 illustrates rat serum calcium levels after oral
administration of calcitonin and calcitonin encapsulated in the
vegetable protein microsphere of the present invention as described
in Example 5.
DETAILED DESCRIPTION OF THE INVENTION
All patents, patent applications, and literatures cited in the
specification are hereby incorporated by reference in their
entirety. In the case of inconsistencies, the present disclosure,
including definitions, will prevail.
The modified vegetable protein microspheres of the present
invention may be prepared by reacting a hydrolyzed vegetable
protein with a chemical modifying agent which reacts with free
amino residues present in the protein. The modified vegetable
protein is then converted into microspheres which encapsulate
active ingredients, e.g. drugs. A number of advantages are
obtainable by the present invention which include (a) the use of
readily available and inexpensive starting materials and (b) a
cost-effective method for preparing and isolating
microsphere-producing modified proteins. The overall modification
process is simple to perform and is amenable to industrial scale-up
production.
According to the method of the present invention, an acid or enzyme
hydrolyzed vegetable protein is useful in practicing the invention.
The vegetable protein generally contains titratable carboxylic acid
groups (COOH) ranging between about 3 and about 8
milliequivalents/g, preferably between about 4 and about 6
milliequivalents/g, total free amino groups (NH.sub.2) ranging
between about 3 and about 9 milliequivalents/g, preferably ranging
between about 4 and about 7 milliequivalents/g NH.sub.2. The
molecular weight of the vegetable protein ranges between about 100
D and about 2000 D, preferably between about 200 and about 500
D.
Hydrolyzed vegetable protein is available from a variety of
commercial sources. Non-limiting examples of such sources include
Ajinomoto USA, Inc. (Teaneck, N.J. 07666, U.S.A.); Central Soya
Co., Inc. (Fort Wayne, Ind., U.S.A.); and Champlain Industries,
Inc. (Clifton, N.J., U.S.A.) and additional companies listed in
"Food Engineering Master", an annual publication of Chilton Co.,
Radnor, Pa. 19089, U.S.A. A particularly preferred hydrolyzed
vegetable protein in practicing this invention is available from
Ajinomoto USA under the tradename AJI-EKI. This product is an acid
hydrolyzed liquid soybean protein which is derived from defatted
soybean meal.
If desired, a dried protein extract of the hydrolyzed vegetable
protein solution may be used to prepare the modified vegetable
protein of the invention. The dried protein extract is preparable
by extracting the hydrolyzed vegetable solution with a suitable
solvent, e.g., methanol, followed by evaporating the solvent
extract.
The vegetable protein is then dissolved in aqueous alkaline
solution of a metal hydroxide, e.g., sodium or potassium hydroxide,
and heated at a temperature ranging between about 50.degree. C. and
about 70.degree. C., preferably between about 50.degree. C. and
about 60.degree. C., for a period ranging between about 10 minutes
and about 40 minutes, preferably about 15 minutes. The amount of
alkali employed per mmole of titratable NH.sub.2 in the vegetable
protein generally ranges between about 2 and about 3 mmole,
preferably between about 2.2 and about 2.5 mmole. The pH of the
solution generally ranges between about 8 and about 13, preferably
ranging between about 9 and about 10.
Thereafter, an amine modifying agent is then added to the reaction
mixture while stirring. The amine modifying agents are compositions
that can react with the free amino (NH.sub.2) residues present in
the protein. Some non-limiting examples of amine modifying agents
useful in practicing the present invention include sulfonating
agents such as benzene sulfonyl chloride and acylating agents such
as benzoyl chloride.
The amount of amine modifying agent in relation to the quantity of
hydrolyzed vegetable protein employed is based on the equivalents
of total free NH.sub.2 in the vegetable protein. Thus, between
about 0.3 and about 1.2 equivalents of modifying agent are used for
each molar equivalent of total NH.sub.2 groups in vegetable
protein, and preferably between about 0.6 and about 1.0 equivalents
of the modifying agent for each molar equivalent of total NH.sub.2
groups in the hydrolyzed vegetable protein.
In practicing the invention, the mixture of vegetable protein and
modifying agent is maintained at a temperature generally ranging
between about 50.degree. C. and about 70.degree. C., preferably
between about 60.degree. C. and about 65.degree. C. for a period
ranging between about 2 and about 5 hours.
The reaction is quenched by adjusting the pH of the mixture with a
suitable acid, e.g., concentrated hydrochloric acid, until the pH
reaches between about 2 and about 3. The mixture separates on
standing at room temperature to form an opaque upper layer and a
dark viscous lower layer. The upper layer is discarded and modified
vegetable protein is collected from the lower layer by filtration.
The crude modified vegetable protein is then dissolved in water at
a pH ranging between about 9 and about 13, preferably between about
11 and about 13. Insoluble materials are removed by filtration and
the filtrate is dried in vacuo. The yield of modified protein
generally ranges between about 30 and about 60%, usually about
45%.
The modified vegetable protein of the present invention is soluble
in alkaline aqueous solution (pH.gtoreq.9.0); partially soluble in
ethanol, n-butanol and 1:1 (v/v) toluene/ethanol solution and
insoluble in neutral water. The titratable functional groups
remaining in the vegetable protein after modification are as
follows: carboxylic acid groups (COOH) ranging between about 1.5
and about 3.5 milliequivalents/g, preferably about 2.3
milliequivalents/g, amino groups (NH.sub.2) ranging between about
0.3 and about 0.9 milliequivalents/g, preferably about 0.5
milliequivalents/g. The molecular weight of the modified vegetable
protein ranges between about 200 D and about 2000 D, preferably
between about 200 D and about 500 D.
The modified vegetable protein of the present invention can be used
immediately to microencapsulate an active pharmacological agent or
the protein can be concentrated or dried by conventional means and
stored for future use.
The modified vegetable protein may be purified by fractionation on
solid column supports such as alumina, using methanol/n-propanol
mixtures as the mobile phase; reverse phase column supports using
trifluoroacetic acid/acetonitrile mixtures as the mobile phase; and
ion exchange chromatography using water as the mobile phase. When
anion exchange chromatography is performed, a subsequent 0-500 mM
sodium chloride gradient is employed. The modified vegetable
protein may also be purified by extraction with a lower alcohol
such as methanol, butanol, or isopropanol to remove low molecular
weight contaminants.
The following procedure may be employed to make microspheres from
purified modified vegetable protein. Modified vegetable protein is
dissolved in deionized water at a concentration ranging between
about 75 and about 200 mg/ml, preferably about 100 mg/ml at a
temperature between about 25.degree. C. and about 60.degree. C.,
preferably about 40.degree. C. Particulate matter remaining in the
solution may be removed by conventional means such as gravity
filtration over filter paper.
Thereafter, the protein solution, maintained at a temperature of
about 40.degree. C., is mixed 1:1 (V/V) with an aqueous acid
solution (also at about 40.degree. C.) having an acid concentration
ranging between about 0.05N and about 2N, preferably about 1.7N.
The resulting mixture is further incubated at 40.degree. C. for a
period of time effective for microsphere formation as observed by
light microscopy. In practicing this invention, the preferred order
of addition is to add the protein solution to the aqueous acid
solution.
Suitable acids include any acid which does not (a) adversely effect
the protein, e.g., chemical decomposition; (b) interfere with
microsphere formation; (c) interfere with microsphere encapsulation
of cargo; and (d) adversely interact with the cargo. Preferred
acids for use in this invention include acetic acid, citric acid,
hydrochloric acid, phosphoric acid, malic acid and maleic acid.
In practicing the invention, a microsphere stabilizing additive
preferably incorporated into the aqueous acid solution or into the
protein solution, prior to the microsphere formation process. The
presence of such additives promotes the stability and
dispersibility of the microspheres in solution.
The additives may be employed at a concentration ranging between
about 0.1 and 5% (W/V), preferably about 0.5% (W/V). Suitable, but
non-limiting, examples of microsphere stabilizing additives include
gum acacia, gelatin, polyethylene glycol, and polylysine.
Under these conditions, the modified vegetable protein molecules
form hollow microspheres of less than 10 microns in diameter. If
the protein microspheres are formed in the presence of a soluble
material, e.g., a pharmaceutical agent in the aforementioned
aqueous acid solution, this material will be encapsulated in the
hollows of the microspheres and confined within the protein wall
defined by the spherical structure. In this way, one can
encapsulate pharmacologically active materials such as peptides,
proteins, and polysaccharides as well as charged organic molecules,
e.g., quinolones or antimicrobial agents, having poor
bioavailability by the oral route. The amount of pharmaceutical
agent which may be encapsulated by the microsphere is dependent on
a number of factors which include the concentration of agent in the
encapsulating solution, as well as the affinity of the cargo for
the carrier.
The modified vegetable protein microspheres of the invention are
pharmacologically harmless and do not alter the physiological and
biological properties of the active agent. Furthermore, the
encapsulation process does not alter the pharmacological properties
of the active agent. While any pharmacological agent can be
encapsulated within the protein microspheres, it is particularly
valuable for delivering chemical or biological agents which
otherwise would be destroyed or rendered less effective by
conditions encountered within the body of the mammal to which it is
administered, before the microsphere reaches its target zone (i.e.,
the area in which the contents of the microsphere are to be
released) and which are poorly absorbed in the gastrointestinal
tract.
The protein microspheres of the invention are particularly useful
for the oral administration of certain pharmacological agents,
e.g., small peptide hormones, which, by themselves, pass slowly or
not at all through the gastro-intestinal mucosa and/or are
susceptible to chemical cleavage by acids and enzymes in the
gastrointestinal tract. Non-limiting examples of such agents
include human or bovine growth hormone, interferon and
interleukin-II, calcitonin, erythropoietin, atrial naturetic
factor, antigens and monoclonal antibodies.
The particle size of the microsphere plays an important role in
determining release of the active agent in the targeted area of the
gastrointestinal tract. Microspheres having diameters between about
.ltoreq.0.1 microns and about 10 microns, preferably between about
5.0 microns and about 0.1 microns, and encapsulating active agents
are sufficiently small to effectively release the active agent at
the targeted area within the gastrointestinal tract. Small
microspheres can also be administered parenterally by being
suspended in an appropriate carrier fluid (e.g., isotonic saline)
and injected into the circulatory system or subcutaneously. The
mode of administration selected will, of course, vary, depending
upon the requirement of the active agent being administered. Large
protein microspheres (>10 microns) tend to be less effective as
oral delivery systems.
The size of the microspheres formed by contacting modified
vegetable protein with water or an aqueous solution containing
active agents can be controlled by manipulating a variety of
physical or chemical parameters, such as the pH, osmolarity or
ionic strength of the encapsulating solution, and by the choice of
acid used in the encapsulating process.
The vegetable protein-derived microspheres of the present invention
are suitable for oral administration of peptide hormones, e.g.,
insulin, and polysaccharides, e.g., heparin, which otherwise would
be quickly destroyed in the stomach. They also are suitable for
protecting the stomach from gastric irritants, such as aspirin and
NSAID'S. When such aspirin containing microspheres are orally
administered, they pass through the gastrointestinal mucosa and
release the aspirin far more rapidly than conventional enterically
coated aspirin, which first must traverse the stomach and then must
enter the bloodstream from the intestine after the enteric coating
has dissolved.
The microspheres of the invention may be orally administered alone
as solids in the form of tablets, pellets, capsules, and granulates
suitable for suspension in liquids such as water or edible oils.
Similarly, the microspheres can be formulated into a composition
containing one or more physiologically compatible carriers or
excipients, and which can be administered via the oral route. These
compositions may contain conventional ingredients such as gelatin,
polyvinylpyrrolidone and fillers such as starch and methyl
cellulose. Alternatively, small microspheres (size less than 10
.mu.m) can be administered via the parenteral route.
The following examples are illustrative of the invention but are
not intended to limit the scope of the invention.
EXAMPLE 1
Modification of Soybean protein with benzenesulfonyl chloride
a. Extraction of soybean protein
3.2 L of acid hydrolyzed liquid soybean protein solution (AJI-EKI,
Ajinomoto USA, Inc.) was reduced in vacuo to give 1440 g of solid
powder. This solid was extracted 3 times with methanol (2 L per
extraction). Methanol was removed from the pooled extracts by
evaporation. The yield of soybean protein as a dark brown powder
was 608 g. The functional groups of the soybean protein powder was
titrated using conventional procedures. See, for example, "A
Laboratory Manual of Analytical Methods of Protein Chemistry," Vol.
1-3, Editors P. Alexander and R. J. Block, Pergamon Press, 1960 and
1961. The soybean protein contained the following functional
groups: 3.7 milliequivalents/g of COOH; 0.44 milliequivalents/g
free N-terminal NH.sub.2 ; and 3.48 milliequivalents/g total free
NH.sub.2. The molecular weight of the soybean protein ranged from
100 to 2000 D.
b. Modification of soybean protein
The dried soybean protein of step (a) (600 g, 2.5 equivalents of
total free NH.sub.2) was dissolved in 3 L of aqueous 2N potassium
hydroxide solution (2.25 mole excess) and the solution was heated
at 60.degree. C. for 30 minutes. Thereafter, benzenesulfonyl
chloride (460 g, 2.60 moles) was added dropwise to the mixture and
the reaction temperature was monitored so that it did not exceed
65.degree. C. The reaction continued, with stirring, for 4 hours at
63.degree. C. The reaction mixture cooled to room temperature, then
acidified to pH 3.0 with 20% aqueous HCl solution and modified
soybean protein precipitated out. The modified soybean protein was
then washed twice with distilled water (1 L) and dissolved in 2N
aqueous sodium hydroxide solution until a pH of 8.5-9 resulted. The
solution was filtered to remove particulates and the filtrate was
reduced and dried in vacuo to give dry modified product (257 g,
yield=24%). The product had the following titratable groups: 2.3
milliequivalents/g of COOH; 0.2 milliequivalents/g N-terminal free
NH.sub.2 ; and 0.3 milliequivalents/g total free NH.sub.2.
EXAMPLE 2
Modification of soybean protein with benzoyl chloride
A commercial hydrolyzed water solution of soybean protein (AJI-EKI,
Ajinomoto USA, Inc.) was used in this Example without further
extraction. The protein in solution contained the following
functional groups: 2.6 milliequivalents/ml of COOH and 2.0
milliequivalents/ml NH.sub.2. The molecular weight of the soybean
protein was approximately 6.5 kD.
To the soybean solution (240 mL, 0.5 equivalents free NH.sub.2) was
added 107 mL of 10N aqueous potassium hydroxide solution followed
by 200 mL of distilled water. The solution was then placed in an
ice bath (5.degree. C.) and benzoyl chloride (70 g, 0.5 moles) was
added dropwise within a temperature range between 10 to 25.degree.
C. The reaction mixture was then stirred for 4.5 hours at room
temperature. The pH of the reaction mixture was then reduced from
13.2 to 2.8 with concentrated HCl. After being allowed to settle
for 1 hour, the precipitated modified soybean protein was collected
by filtration and washed with distilled water. The soybean protein
was then dissolved in 2N aqueous sodium hydroxide solution to give
a solution (pH 12.6) which was then evaporated to afford 48 g of
dried product (% yield=41%).
EXAMPLE 3
Preparation of Empty Microspheres With Modified Soybean Protein
This Example illustrates a method for the preparation and cleaning
of empty modified soybean protein microspheres.
Procedure
1. Reagents:
a. Modified protein powder prepared as described in Example 1
b. Anhydrous citric acid (USP)
c. Gum acacia NF
d. Deionized water
e. Glacial acetic acid
2. Equipment
a. pH meter
b. Eppendorf pipette (0-100 ul) and tips
c. Water bath, 40.degree. C.
d. liquid nitrogen
e. lyophilization flasks
3. Preparation of Solutions:
a. Protein solution--Dissolve 100 mg modified soybean protein in ml
deionized water (or multiples thereof). Filter through a Whatman #1
filter paper (if necessary) and keep at 40.degree. C. in a water
bath. This is solution A.
b. 1.7N citric acid with 0.5% acacia--Dissolve 5 g of acacia and
109 g of citric acid in 1 liter deionized water. Incubate at
40.degree. C. This is solution B.
4. Preparation of Microspheres:
a. Add all of solution A to solution B rapidly in one step while
swirling solution B by hand, in a 40.degree. C. water bath.
EXAMPLE 4
Preparation of Soybean protein Microsphere containing Encapsulated
Heparin
This Example describes a method for the preparation and cleaning of
heparin microspheres.
Procedure
1. Reagents:
a. Modified protein powder prepared as described in Example 1
b. Heparin
c. Anhydrous citric acid (USP)
d. Gum acacia NF
e. Deionized water
f. Desiccant
g. Liquid nitrogen
2. Equipment:
a. Magnetic stirrer
b. Buret
c. Microscope
d. Clinical centrifuge
e. Dialysis membrane tubing (Spectrum 6, 10 mm, 50,000 M. W.
Cutoff)
f. pH meter
g. Lyophilizer (Labconco #75035)
h. Lyophilizing flasks (150-300 mL)
i. Rotating shell freezer
j. Isopropanol/dry ice bath or liquid N.sub.2
k. Mortar and pestle
l. Storage containers (500 mL)
m. Eppendorf piper (0-100 uL)
n. Plastic closures for dialysis tubing (Spectrum)
o. 2 mL syringe with 0.45 .mu.m Acrodisk
3. Preparation of Solutions:
a. Protein Solution A* (80 mg/ml): Add 160 mg of modified soybean
protein and dissolve to 1 ml with deionized water. Using a 2 ml
syringe filter through a 0.45 .mu.m Acrodisk into a 10 ml test
tube. Keep at 40.degree. C.
b. Solution B (1.7N citric acid with 1% gum): Dissolve 10 g of
acacia and 109 g of citric acid in 1
c. Solution C (Heparin solution): Dissolve heparin in Solution B at
150 mg/mL and keep at 40.degree. C.
4. Preparation of Microspheres:
a. Add all of solution A to solution C quickly while swirling
solution C slowly, by hand, in a 40.degree. C. water bath.
5. Cleaning of Microspheres:
a. Transfer the suspension with a syringe (no needle) to dialysis
tubing and seal with plastic closures. Tubing should be no more
than 70% full.
b. Discard any amorphous material sedimented and/or aggregated on
the surface.
c. Dialyze the microsphere suspension against acetic acid (using 20
mL of acetic acid solution per ml of microsphere suspension) while
stirring the acetic acid solution with a magnetic stirrer.
d. Replace the acetic acid solution every hour. Continue dialyzing
for a total of 3 hours.
6. Lyophilization:
a. Add one part of 50% Trehalose (Sigma) into nine parts of
dialyzed microsphere solution. Flash freeze microspheres in a
freeze-drying flask using the shell freezer adjusted to rotate at
ca. 190 rpm and immersed in a liquid nitrogen bath.
b. Freeze dry for 24 hours or until dry as evidenced by lack of
self-cooling.
c. Record weight of dry microspheres.
d. Grind to a fine powder with mortar and pestle.
e. Transfer to amber container, seal with desiccant, and store at
room temperature.
7. Resuspension:
a. Weigh the lyophilized powder and calculate the amount of protein
in the powder.
b. Add 0.85N citric acid into the lyophilized powder at 40.degree.
C. The final concentration of protein is 80 mg/ml.
EXAMPLE 5
Evaluation of Insulin Microspheres in Rats
In this Example, the insulin microspheres prepared in accordance
with Example 3 were evaluated in rats. Twelve rats were divided
into two groups as follows:
1. oral insulin microspheres: 3 mg insulin/kg body weight by oral
garage (six rats);
2. Raw insulin (no encapsulation): 3 mg insulin/kg body weight by
oral garage (six rats).
Oral garage dosing of rats was performed. Insulin microspheres were
prepared immediately prior to dosing and Group 1 rats each receive
an appropriate dosage of the microsphere suspension. Group 2 rats
received the unencapsulated insulin. Approximately 0.5 ml of blood
was withdrawn from each rat just prior to dosing ("0" time) and 1
to 6 h post-dosing. Serum from the blood samples were stored at
-20.degree. C.
The glucose levels of thawed serum taken from the rats were
analyzed by conventional methods. As shown in FIG. 1, sharp
decreases in serum glucose levels were observed in groups 1 rats
receiving the encapsulated insulin. In contrast, the serum glucose
levels in group 2 rats slightly increased from t=0. The results
show that encapsulated insulin had a greater biological effect,
when administered orally, in contrast to unencapsulated
insulin.
EXAMPLE 6
Preparation of Microsphere Encapsulated Calcitonin
Encapsulation of salmon calcitonin in soybean protein microspheres
were performed in the same manner described in Example 3.
Calcitonin was obtained from Sandoz (Basil, Switzerland) and a 150
mg/mL calcitonin solution in 1.7N citric acid solution with 1% gum
was prepared as described in Example 3.
EXAMPLE 7
Evaluation of Calcitonin Microspheres in Rats
In this Example, the calcitonin microspheres prepared in accordance
with Example 5 were evaluated in rats. Twelve rats were divided
into two groups as follows:
1. oral calcitonin microspheres: 60 .mu.g calcitonin/kg body weight
by oral garage (six rats).
2. oral unencapsulated microspheres: 60 .mu.g calcitonin/kg body
weight by oral gavage (3 rats) (Control).
Oral garage dosing of rats was performed. Calcitonin microspheres
were prepared immediately prior to dosing and Group 1 rats received
an appropriate dosage of the microsphere suspension. Group 2 rats
received the unencapsulated calcitonin. Approximately 0.5 ml of
blood was withdrawn from each rat just prior to dosing ("0" time)
and 1 h, 2 h and 3 h post-dosing. Serum from the blood samples were
stored at -20.degree. C.
The calcium levels of thawed serum taken from group 1 and 2 rats
were analyzed by conventional methods. As shown in FIG. 2, sharp
decreases in serum calcium levels were observed in group 1 rats
receiving the encapsulated calcitonin. In contrast, the calcium
levels in group 2 rats slightly decreased from t=0. The results
show that encapsulated calcitonin had a greater biological effect,
when administered orally, in contrast to unencapsulated
calcitonin.
* * * * *